It is now well known that cells can be infected by HIV through a process by which fusion occurs between the cellular membrane and the viral membrane. The generally accepted model of this process is that the viral envelope glycoprotein complex (gp120/gp41) interact with cell surface receptors on the membranes of the target cells. Following binding of gp120 to cellular receptors (e.g., CD4 in combination with a chemokine co-receptor such as CCR-5 or CXCR-4), induced is a conformational change in the gp120/gp41 complex that allows gp41 to insert into the membrane of the target cell and mediate membrane fusion.
The amino acid sequence of gp41, and its variation among different strains of HIV, is well known. FIG. 1 is a schematic representation of the generally accepted functional domains of gp41 (note the amino acid sequence numbers may vary slightly depending on the HIV strain). The fusion peptide (fusogenic domain) is believed to be involved in insertion into and disruption of the target cell membrane. The transmembrane domain, containing the transmembrane anchor sequence, is located at the C-terminal end of the protein. Between the fusion peptide and transmembrane anchor are two distinct regions, known as heptad repeat (HR) regions, each region having a plurality of heptads. One region, HR1, nearer to the N-terminal end of the protein, has been generally described as comprising amino acid residues from about 545 to about 595 of the amino acid sequence of gp160 (SEQ ID NO:1). However, the amino acid numbering of gp160 depends on the strain from which the amino acid sequence was deduced. The amino acid sequence comprising the HR1 region and the amino acid sequence comprising the HR2 region are each highly conserved regions in the HIV-1 envelope protein (Shu et al., 1999, Biochemistry, 38:5378–5385; Hanna et al., 2002, AIDS 16:1603–8). The HR2 region, nearer to the C-terminal end of the protein as compared to the HR1 region, has been generally described as comprising amino acids in the positions from about 628 to about 678 of the amino acid sequence of gp160 (SEQ ID NO:2). The HR regions share structural and functional features. For example, each HR region has a plurality of 7 amino acid residue stretches or “heptads” (the 7 amino acids in each heptad designated “a” through “g”), wherein the amino acids in the “a” position and “d” position are generally hydrophobic. Also present in each HR region is one or more leucine zipper-like motifs (also referred to as “leucine zipper-like repeats”) comprising an 8 amino acid sequence initiating with and ending with an isoleucine or leucine. Most frequently, the HR2 region has just one leucine zipper like-motif, whereas the HR1 region has five leucine zipper-like motifs. The heptads and leucine zipper-like motifs each contribute to formation of a coiled coil structure of gp41 and of a coiled coil structure of peptides derived from the HR regions. Generally, coiled coils are known to be comprised of two or more helices that wrap around each other in forming oligomers, with the hallmark of coiled coils being a heptad repeat of amino acids with a predominance of hydrophobic residues at the first (“a”) and fourth (“d”) positions, charged residues frequently at the fifth (“e”) and seventh (“g”) positions, and with the amino acids in the “a” position and “d” position being determinants that influence the oligomeric state and strand orientation (see, e.g., Akey et al., 2001, Biochemistry, 40:6352–60).
It was discovered that synthetic peptides derived from either the HR1 region (“HR1 peptides”) or HR2 region (“HR2 peptides”) of HIV gp41 inhibit transmission of HIV to host cells both in in vitro assays and in in vivo clinical studies (see, e.g., Wild et al., 1994, Proc. Natl. Acad. Sci. USA, 91:9770–9774; U.S. Pat. Nos. 5,464,933 and 5,656,480 licensed to the present assignee; and Kilby et al., 1998, Nature Med. 4:1302–1306). More particularly, HR1 peptides as exemplified by DP107 (also known as T21; SEQ ID NO:3) blocked infection of T cells with 50% effective concentration values (EC50) of 1 μg/ml (see, e.g., Lawless et al., 1996, Biochemistry, 35:13697–13708). HR2 peptides, as exemplified by DP178 (also known as T20; SEQ ID NO:4) blocked infection of T cells with 50% effective concentration values (EC50) in the ng/ml range. Pioneering potent synthetic peptides, which comprise one or more enhancer sequences linked to a core HIV gp41 amino acid sequence, inhibit HIV membrane fusion and thereby prevent transmission of the virus to a host cell, have been described previously (see, e.g., U.S. Pat. Nos. 6,258,782 and 6,348,568 assigned to the present assignee). Currently, like other peptides known in the art, to most efficiently deliver an amount effective for antiviral activity, such synthetic peptides are administered frequently (e.g., daily injections) to attain and maintain a level in the bloodstream sufficient for such a therapeutic effect. Additionally, injection site reactions are the most common adverse event in individuals receiving the currently available injectable solution formulations of HIV fusion inhibitors. For example, in one Phase III study of T20 administered subcutaneously, and using a mannitol-based formulation (without a polyol as taught in the present invention), injection site reactions (manifested by one or more of reddening, swelling, and discomfort at the injection site) were experienced by 98% of patients receiving treatment, with 3.3% of patients citing such reactions as a reason for discontinuing treatment. Another limitation with the current injectable solution formulation of HIV fusion inhibitors is that it is difficult to achieve a desirable injectable solution containing a concentration of synthetic peptide of no less than 100 mg/ml without problems of viscosity (where the formulation resembles a gel rather than a solution) and/or instability (e.g., precipitation, over a predetermined time period, of synthetic peptide out of solution).
Polyols, particularly polyethylene glycol (PEG), have been found to be well tolerated and are believed to have a relatively low level of toxicity when used as a pharmaceutically acceptable carrier in an injectable solution of a drug formulation. Thus, PEG has been used as a pharmaceutically acceptable carrier in a number of regulatory-approved injectable solution formulations that contain drug comprising chemical compositions other than peptides and proteins. The amount of PEG in such a formulation is typically present in an amount from about 0.1 weight percent to about 5 weight percent of the formulation. PEG has not been used as a pharmaceutically acceptable carrier for maintaining proteins and peptides in solution, but rather has been used in the precipitation of proteins and peptides. For example, hepatitis B surface antigen protein may be purified using a cycle of precipitation with from 1% to 10% PEG (w/v; see, e.g., U.S. Pat. No. 5,462,863); secretory IgA can be precipitated with PEG at a concentration of 15 to 25 weight per volume (w/v) percent of PEG; fibrinogen can be precipitated with PEG amounting to 2.5% by weight; asparaginase can be precipitated with a solution of 40 to 60 weight percent PEG, and antihemophilic factor may be precipitated at a final concentration of 3 percent to 6 percent PEG (w/v). Thus, one concern in using PEG at concentrations equal to or greater than 5 weight percent in a pharmaceutical composition as a pharmaceutically acceptable carrier is precipitation out of solution of the protein or peptide that is to be administered in an injectable solution formulation, a very undesirable effect. In one instance (see, e.g., U.S. Pat. No. 6,004,549), disclosed is a pharmaceutical composition comprised of a suspension of a protein in a polyol; i.e., a crystalline form of interferon suspended in a solution or gel containing 40% aqueous solution of PEG8000 (w/v) or a 50% solution of PEG 3350 (the number following “PEG” is approximate molecular size in daltons of the PEG referenced, as will be discussed in more detail herein).
However, a pharmaceutical composition comprised of a solution comprised of mixture of synthetic peptide (HIV fusion inhibitor) and a polyol such as PEG in a final concentration of no less than 5 weight percent (%) (e.g., weight/volume percent) and no more than 75 weight %, has not heretofor been disclosed. Until the discovery of the present invention there remained a long-felt need in the art for a formulation of a pharmaceutical composition which can (a) be used as an injectable solution, (b) contain synthetic peptide (HIV fusion inhibitor) in a concentration of no less than 100 mg/ml in a solution having sufficient stability for use for its intended purpose, and additionally, in possibly reducing the number of injections needed to administer an effective amount of synthetic peptide for achieving a therapeutic effect, and (c) which may minimize an injection site reaction. The present invention addresses these needs.